辐射诱导树突状细胞促进Treg分化及肺上皮间充质转化

目的探讨树突状细胞经60Co-γ射线照射后激活调节性T淋巴细胞(regulatory T cells,Treg)的能力,以及在Treg诱导的上皮间充质转化(epithelial-mesenchymal transition,EMT)中的作用。方法免疫磁珠分选的小鼠CD4+CD25-常规T细胞(conventional T cells,Tcon)与60Co-γ线照射(6 Gy)后的树突状细胞共培养,72 h后采用流式术检测CD4+CD25+Treg细胞的比例。通过磁珠分选后的Treg与小鼠肺上皮细胞系MLE-12共培养72 h后取MLE-12细胞,免疫荧光和Western印迹技术检测上皮标志蛋白E-钙黏蛋白(E-cadherin,E-cad)和细胞表面蛋白C(prosurfactant protein C,Spc)以及间充质标志蛋白N-钙黏蛋白(N-cadherin,N-cad)和波形蛋白(vimentin)的表达。结果免疫磁珠法分选小鼠脾Treg比例>90%,效果良好;树突状细胞照后抗原提呈功能增强,能显著诱导Tcon分化为Treg; Western印迹和免疫荧光实验证明,Treg能明显促进上皮标志E-cad和Spc表达下调,间充质标志N-cad和vimentin表达升高。结论树突状细胞照射后能显著诱导Treg分化,而Treg能明显诱导肺上皮细胞发生EMT。

Thrombin promotes human lung fibroblasts to proliferate via NADPH oxidase/reactive oxygen species/extracellular regulated kinase signaling pathway

Background Thrombin is a multifunctional serine protease that plays a crucial role in hemostasis following tissue injury.In addition to its procoagulation effect, thrombin is also a potent mesenchymal cell mitogen, therefore it plays important roles in the local proliferation of mesenchymal cells in the tissue repair process. Reactive oxygen species （ROS） can induce some human cells to proliferate at lower rates while at higher concentrations they promote cells to undergo apoptosis or necrosis. Accumulative evidence suggests that thrombin can induce some cells to produce ROS. Based on these observations, we provide a hypothesis that thrombin can stimulate human lung fibroblasts to produce ROS, which play an important role in human lung fibroblast proliferation.Methods ROS were detected in fibroblasts at 30 minutes and 60 minutes following thrombin （20 U/ml） exposure using flow cytometry. The ratio of reduced glutathione/oxidized glutathione （GSH/GSSG） was assayed in lung fibroblasts using a commercial kit following treatment with thrombin at different concentrations. NADPH oxidase and the extracellular regulated kinase1/2 （ERK1/2） signaling pathway were detected by Western blotting after thrombin stimulation to lung fibroblasts.Results Thrombin, at 20 U/ml, stimulated human lung fibroblasts （HLF） to generate ROS in a time dependent manner.The ratio of GSH/GSSG in fibroblasts treated with thrombin showed a significant decrease. NADPH oxidase was activated and the ERK1/2 signal pathway was involved in the proliferation process of fibroblasts treated with thrombin.Conclusion The activation of NADPH oxidase by thrombin leads to the production of ROS, which promotes fibroblasts proliferation via activation of the ERK1/2 signaling pathway.